U.S. patent application number 09/965976 was filed with the patent office on 2002-08-08 for topoisomerase inhibitors.
Invention is credited to Bachand, Carol, Balasubramanian, Neelakantan, Beaulieu, Francis, Frennesson, David B., Mahler, Mikael, Ruediger, Edward H., Saulnier, Mark G..
Application Number | 20020107237 09/965976 |
Document ID | / |
Family ID | 22899064 |
Filed Date | 2002-08-08 |
United States Patent
Application |
20020107237 |
Kind Code |
A1 |
Saulnier, Mark G. ; et
al. |
August 8, 2002 |
Topoisomerase inhibitors
Abstract
The present invention relates to novel N12, N13-bridged sugar
derivatives of indolylopyrrolocarbazoles and pharmaceutical
formulations thereof which exhibit topoisomerase-I activity and are
useful in inhibiting the proliferation of tumor cells.
Inventors: |
Saulnier, Mark G.;
(Higganum, CT) ; Ruediger, Edward H.; (Greenfield
Park, CA) ; Balasubramanian, Neelakantan; (Madison,
CT) ; Mahler, Mikael; (Outremont, CA) ;
Beaulieu, Francis; (Laprairie, CA) ; Bachand,
Carol; (Candiac, CA) ; Frennesson, David B.;
(Naugatuck, CT) |
Correspondence
Address: |
STEPHEN B. DAVIS
BRISTOL-MYERS SQUIBB COMPANY
PATENT DEPARTMENT
P O BOX 4000
PRINCETON
NJ
08543-4000
US
|
Family ID: |
22899064 |
Appl. No.: |
09/965976 |
Filed: |
September 27, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60238726 |
Oct 6, 2000 |
|
|
|
Current U.S.
Class: |
514/211.08 ;
514/219; 540/545; 540/556 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 35/00 20180101; C07H 19/23 20130101; C07H 9/06 20130101 |
Class at
Publication: |
514/211.08 ;
514/219; 540/545; 540/556 |
International
Class: |
A61K 031/553; A61K
031/551; C07D 498/16 |
Claims
What is claimed is:
1. A compound of Formula (I) or pharmaceutically acceptable salt or
solvate thereof, useful for inhibiting topoisomerase I and the
proliferation of tumor cells 37wherein Z is selected from the group
consisting of Formula (A), Formula (B), Formula (C), Formula (D),
Formula (E), Formula (F) and Formula (G) 38R is hydrogen, OH,
OC.sub.1-7alkyl, NH.sub.2, N(C.sub.1-3alkyl).sub.2, or
C.sub.1-7alkyl, wherein said C.sub.1-7alkyl or C.sub.1-3alkyl is
optionally substituted with one or more substituents selected from
the group consisting of halogen, CN, SR.sup.9, OR.sup.9 and
NR.sup.9R.sup.10; R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5
are each independently selected from the group consisting of
hydrogen, C.sub.1-7alkyl, C.sub.3-7cycloalkyl, halogen, azido,
NR.sup.9R.sup.10, NHC(O)NR.sup.9R.sup.10, NHC(O)OR.sup.9,
C(O)OR.sup.9, SR.sup.9 and OR.sup.9, wherein said C.sub.1-7alkyl is
optionally substituted with one or more substituents selected from
the group consisting of halogen, CN, SR.sup.9, OR.sup.9 and
NR.sup.9R.sup.10; provided that no more than two of the variables
selected from the group consisting of R.sup.1, R.sup.2, R.sup.3,
R.sup.4 and R.sup.5 may be C.sub.3-7cycloalkyl, azido,
NHC(O)NR.sup.9R.sup.10 or NHC(O)OR.sup.9; R.sup.7 and R.sup.8 are
independently OH or H or R.sup.7 and R.sup.8 together form .dbd.O;
R.sup.9 and R.sup.10 are independently selected from the group
consisting of hydrogen, C.sub.1-7alkyl and C.sub.3-7cycloalkyl,
wherein said C.sub.1-7alkyl is optionally substituted with one or
more substituents selected from the group consisting of halogen,
CN, OH, O--C.sub.1-7alkyl, NH.sub.2 and N(C.sub.1-3alkyl).sub.2; or
R.sup.9 and R.sup.10 together with the nitrogen atom to which they
are attached form a non-aromatic 5-8 membered heterocycle
containing one or two of the same or different heteroatoms selected
from the group consisting of O, N and S; m is 0 or 1; and X.sup.1,
X.sup.1', X.sup.2 and X.sup.2' are independently selected from the
group consisting of hydrogen, halogen, cyano, OR.sup.9, --CF.sub.3,
alkylcarbonyl, C-.sub.1-7alkyl, nitro, alkoxyaminoalkyl,
NR.sup.9R.sup.10, SR.sup.9 and C(O)OR.sup.9; wherein said
C.sub.1-7alkyl is optionally substituted with one or more
substituents selected from the group consisting of halogen, CN,
OR.sup.9, SR.sup.9 and NR.sup.9R.sup.10.
Description
RELATED APPLICATIONS
[0001] This application claims priority benefit under Title 35
.sctn.119(e) of U.S. provisional Application No. 60/238,726, filed
Oct. 6, 2000. The contents of which are herein incorporated by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to sugar derivatives of
indolopyrrolocarbazoles which exhibit topoisomerase I activity and
are useful in inhibiting the proliferation of tumor cells.
BACKGROUND
[0003] Topoisomerases are vital nuclear enzymes which function to
resolve topological dilemmas in DNA, such as overwinding,
underwinding and catenation, which normally arise during
replication, transcription and perhaps other DNA processes. These
enzymes allow DNA to relax by forming enzyme-bridged strand breaks
that act as transient gates or pivotal points for the passage of
other DNA strands. Topoisomerase-targeting drugs appear to
interfere with this breakage-reunion reaction of DNA
topoisomerases. In the presence of topoisomerase active agents, an
aborted reaction intermediate, termed a `cleavable complex`,
accumulates and results in replication/transcription arrest, which
ultimately leads to cell death.
[0004] The development of topoisomerase I active agents therefore
offers a new approach to the multi-regimental arsenal of therapies
currently used in the clinic for the treatment of cancer. An
article in Cancer Chemother. Pharmacol [1994, 34 (suppl): S 41-S
45] discusses topoisomerase I active compounds that are in clinical
studies and these have been found to be effective clinical
anti-tumor agents. Structurally these clinical candidates are
related to the alkaloid camptothecin.
[0005] Indolo[2,3-a]carbazole derivatives related to the
Rebeccamycin class are disclosed (EP Appl. 0 545 195 B1 and
0,602,597 A2; Cancer Research 1993, 53, 490-494; ibid 1995, 55,
1310-1315) and claimed to exhibit anti-tumor activity; however the
major mechanism of action of these derivatives may not be like
camptothecin, which acts as a topoisomerase I poison.
[0006] Indolo[2,3-a]carbazole alkaloids such as rebeccamycin (U.S.
Pat. Nos. 4,487,925 and 4,552,842) and its water-soluble,
clinically-active analog, 6-(2-diethylaminoethyl)rebeccamycin (U.S.
Pat. No. 4,785,085), are useful antitumor agents which target DNA.
Furthermore, fluoroindolocarbazoles such as described in WO
98/07433 are antineoplastic agents with topoisomerase I inhibitory
activity. Indolocarbazoles are also disclosed (WO 9530682) and
claimed to exhibit antitumor activity. Hudkins, et al. have
disclosed a series of fused pyrrolocarbazoles (WO 96/11933 and U.S.
Pat. No. 5,475,110) and showed in vitro biological data such as
inhibition of neuronal choline acetyltransferase (ChAT) and protein
kinase C (PKC) inhibition for some compounds. U.S. Pat. No.
5,468,849 discloses certain fluororebeccamycin analogs as useful
antitumor agents, along with a process for their production by
fluorotryptophan analog feeding of a rebeccamycin-producing strain
of Saccharothrix aerocolonigenes, preferably Saccharothrix
aerocolonigenes C38,383-RK2 (ATCC 39243). Glicksman, et al disclose
indolocarbazole alkaloids (U.S. Pat. No. 5,468,872) which are
different in structure from those of the present invention. Kojiri,
et al. disclose indolopyrrolocarbazoles having a dissacharide
substituent (WO 96/04293). Weinreb, et al. (Heterocycles 1984, 21,
309) and Kleinschroth, et al. (U.S. Pat. No. 5,043,335) have
disclosed indolopyrrolocarbazole derivatives with a bridging furan
moiety and McCombie, et al. (Bioorg. Med. Chem. Lett. 1993, 3,
1537) have reported a more functionalized bridged furan. Wood, et
al. have reported the total synthesis of (+)-K252a (J. Am. Chem.
Soc. 1995, 117, 10413), a related, naturally-occurring
indolocarbazole alkaloid which has demonstrated PKC inhibitory
activity. During the course of their total synthesis of (+)-K252a,
Fukuyama, et al. (J. Am. Chem. Soc. 1999, 121, 6501) have also
described the isolation of a cycloglycoside as an undesired
product.
[0007] Danishefsky, et al., during the course of their first total
synthesis of staurosporine (J. Am. Chem. Soc. 1996, 118, 2825),
describe the synthesis of an intermediate N12, N13-bridged
indolopyrrolocarbazole. Indolocarbazole derivatives with the
nitrogens linked by a three-atom bridge have been reported to be
potent PKC inhibitors (S. F. Vice, et al. Bioorg. Med. Chem. Lett.
1994, 4, 1333). The synthesis of simple indolocarbazole derivatives
with C1', C-5'-bridging or C1', C3'-bridging glycosides have also
been reported in the literature (B. M. Stolz, J. L. Wood
Tetrahedron Lett. 1995, 36, 8543 and B. B. Shankar, S. W. McCombie
Tetrahedron Lett. 1994, 35, 3005, respectively). Prudhomme, et al.
disclose a series of antitumor indolocarbazoles derived from
rebeccamycin which exhibit a carbohydrate attached to the two
indole nitrogens, and reported their cytotoxicity and their
topoisomerase I and PKC inhibitory activities to be in the
millimolar to micromolar range (Bioorg. Med. Chem. 1998, 6, 1597).
Despite these examples, there remains a need for novel and potent
cytotoxic compounds useful for inhibiting topoisomerase I
activity.
SUMMARY OF THE INVENTION
[0008] Thus according to a first embodiment of the first aspect of
the present invention are provided compounds of Formula (I) and
pharmaceutically acceptable salts and solvates thereof, useful for
inhibiting topoisomerase I and the proliferation of tumor cells
1
[0009] wherein
[0010] Z is selected from the group consisting of Formula (A),
Formula (B), Formula (C), Formula (D), Formula (E), Formula (F) and
Formula (G) 2
[0011] R is hydrogen, OH, OC.sub.1-7alkyl, NH.sub.2,
N(C.sub.1-3alkyl).sub.2, or C.sub.1-7alkyl, wherein said
C.sub.1-7alkyl or C.sub.1-3alkyl is optionally substituted with one
or more substituents selected from the group consisting of halogen,
CN, SR.sup.9, OR.sup.9 and NR.sup.9R.sup.10;
[0012] R.sub.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are each
independently selected from the group consisting of hydrogen,
C.sub.1-7alkyl, C.sub.3-7cycloalkyl, halogen, azido,
NR.sup.9R.sup.10, NHC(O)NR.sup.9R.sup.10, NHC(O)OR.sup.9,
C(O)OR.sup.9, SR.sup.9 and OR.sup.9, wherein said C.sub.1-7alkyl is
optionally substituted with one or more substituents selected from
the group consisting of halogen, CN, SR.sup.9, OR.sup.9 and
NR.sup.9R.sup.10;
[0013] provided that no more than two of the variables selected
from the group consisting of R.sup.1, R.sup.2, R.sup.3, R.sup.4 and
R.sup.5 may be C.sub.3-7cycloalkyl, azido, NHC(O)NR.sup.9R.sup.10
or NHC(O)OR.sup.9;
[0014] R.sup.7 and R.sup.8 are independently OH or H or R.sup.7 and
R.sup.8 together form .dbd.O;
[0015] R.sup.9 and R.sup.10 are independently selected from the
group consisting of hydrogen, C.sub.1-7alkyl and
C.sub.3-7cycloalkyl, wherein said C.sub.1-7alkyl is optionally
substituted with one or more substituents selected from the group
consisting of halogen, CN, OH, O--C.sub.1-7alkyl, NH.sub.2 and
N(C.sub.1-3alkyl).sub.2; or
[0016] R.sup.9 and R.sup.10 together with the nitrogen atom to
which they are attached form a non-aromatic 5-8 membered
heterocycle containing one or two of the same or different
heteroatoms selected from the group consisting of O, N and S;
[0017] m is 0 or 1; and
[0018] X.sup.1, X.sup.1', X.sup.2 and X.sup.2' are independently
selected from the group consisting of hydrogen, halogen, cyano,
OR.sup.9, --CF.sub.3, alkylcarbonyl, C-.sub.1-7alkyl, nitro,
alkoxyaminoalkyl, NR.sup.9R.sup.10, SR.sup.9 and C(O)OR.sup.9;
wherein said C.sub.1-7alkyl is optionally substituted with one or
more substituents selected from the group consisting of halogen,
CN, OR.sup.9, SR.sup.9 and NR.sup.9R.sup.10.
[0019] According to a first embodiment of the first aspect of the
present invention are provided compounds of Formula (I) wherein Z
is selected from the group consisting of Formula (A), Formula (C)
and Formula (D).
[0020] According to another embodiment of the first aspect of the
present invention are provided compounds of Formula (I) wherein Z
is formula (A).
[0021] According to another embodiment of the first aspect of the
present invention are provided compounds of Formula (I) wherein Z
is formula (B).
[0022] According to another embodiment of the first aspect of the
present invention are provided compounds of Formula (I) wherein Z
is formula (C).
[0023] According to another embodiment of the first aspect of the
present invention are provided compounds of Formula (I) wherein Z
is formula (D).
[0024] According to another embodiment of the first aspect of the
present invention are provided compounds of Formula (I) wherein m
is 1.
[0025] According to another embodiment of the first aspect of the
present invention are provided compounds of Formula (I) wherein m
is 0.
[0026] According to another embodiment of the first aspect of the
present invention are provided compounds of Formula (I) wherein R
is hydrogen.
[0027] According to another embodiment of the first aspect of the
present invention are provided compounds of Formula (I) wherein
R.sup.7 and R.sup.8 together are .dbd.O.
[0028] According to another embodiment of the first aspect of the
present invention are provided compounds of Formula (I) wherein
R.sup.7 and R.sup.8 are each H.
[0029] According to another embodiment of the first aspect of the
present invention are provided compounds of Formula (I) wherein
X.sup.2' and X.sup.2 are each F and X.sup.1 and X.sup.1' are each
H.
[0030] According to another embodiment of the first aspect of the
present invention are provided compounds of Formula (I) wherein
X.sup.2 is F and X.sup.2', X.sup.1 and X.sup.1' are each H.
[0031] According to another embodiment of the first aspect of the
present invention are provided compounds of Formula (I) wherein
X.sup.2' is F and X.sup.2, X.sup.1 and X.sup.1' are each H.
[0032] According to another embodiment of the first aspect of the
present invention are provided compounds of Formula (I) wherein
X.sup.2', X.sup.2, X.sup.1 and X.sup.1' are each F.
[0033] According to another embodiment of the first aspect of the
present invention are provided compounds of Formula (I) wherein
X.sup.2' and X.sup.2 are each H and X.sup.1 and X.sup.1' are each
F.
[0034] According to another embodiment of the first aspect of the
present invention are provided compounds of Formula (I) wherein
R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are independently
selected from the group consisting of H, F, OR.sup.9 wherein
R.sup.9 is hydrogen or methyl.
[0035] Other embodiments of the first aspect of the present
inventinon provide compounds of Formula (I) comprising two or more
of the above embodiments of the first aspect suitably combined.
[0036] Embodiments of a second aspect of the present invention
provide a method for inhibiting tumor growth in a mammalian host,
particularly a human host, comprising the administration to said
host of a tumor-growth inhibiting amount of a compound of the
present invention, as defined herein.
[0037] Embodiments of a third aspect of the present invention
provide a method for inhibiting tumor growth in a mammalian host
comprising the administration to said host of a tumor-growth
inhibiting amount of a pharmaceutical formulation of a compound of
the present invention, as defined in the emodiments of the first
aspect of the invention.
[0038] Other embodiments and aspects of the invention will be
apparent according to the description provided below.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The description of the invention herein should be construed
in congruity with the laws and principals of chemical bonding. An
embodiment or aspect which depends from another embodiment or
aspect, will describe only the variables having values and provisos
that differ from the embodiment or aspect from which it depends.
Thus, for example, an embodiment which reads "the compound of
formula (I) according to the n.sup.th aspect of the invention,
wherein R is NH.sub.2" should be read to include all remaining
variables with values defined in the nth aspect and should be read
to further include all the provisos, unless otherwise indicated,
pertaining to each and every variable in the n.sup.th aspect. Where
a variable is defined as having a value of zero, it is understood
that the bond attached to said variable should be removed. For
example, if n=0 and R--X--V.sub.n wherein n can be 0 or 1, then it
is understood that the structure described is R--X not R--X--. The
numbers in the subscript after the symbol "C" define the number of
carbon atoms a particular group can contain. For example
"C.sub.1-7alkyl" means a straight or branched saturated carbon
chain having from one to seven carbon atoms including without
limitation groups such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, t-butyl, n-pentyl, sec-pentyl,
isopentyl, n-hexyl and n-heptyl. "Aryl" means an aromatic
hydrocarbon having from six to ten carbon atoms; examples include
phenyl and naphthyl. "Substituted aryl" or "substituted aralkyl"
means an aryl or aralkyl group independently substituted with one
to five (but particularly one to three) groups selected from the
group consisting of C.sub.1-6alkanoyloxy, hydroxy, halogen,
C.sub.1-6 alkyl, trifluoromethyl, C.sub.1-6alkoxy,
C.sub.2-6alkenyl, C.sub.1-6alkanoyl, nitro, amino, cyano, azido,
C.sub.1-6 alkylamino, and amido. The term "halogen" includes
fluoro, chloro, bromo and iodo.
[0040] It is to be understood that the present invention includes
any and all possible stereoisomers, geometric isomers,
diastereoisomers, enantiomers and anomers, unless a particular
description specifies otherwise.
[0041] The compounds of this invention can exist in the form of
pharmaceutically acceptable salts. Such salts include addition
salts with inorganic acids such as, for example, hydrochloric acid
and sulfuric acid, and with organic acids such as, for example,
acetic acid, citric acid, methanesulfonic acid, toluenesulfonic
acid, tartaric acid and maleic acid. Further, in case the compounds
of this invention contain an acidic group, the acidic group can
exist in the form of an alkali metal salt such as, for example, a
potassium salt and a sodium salt; an alkaline earth metal salts
such as, for example, a magnesium salt and a calcium salt; and
salts with organic bases, such as a triethylammonium salt and an
arginine salt. The compounds of the present invention may be
hydrated or non-hydrated.
[0042] The compounds of this invention can be administered in such
oral dosage forms as tablets, capsules (each of which includes
sustained release or timed release formulations), pills, powders,
granules, elixirs, tinctures, suspensions, syrups and emulsions.
The compounds of this invention may also be administered
intravenously, intraperitoneally, subcutaneously, or
intramuscularly, all using using dosage forms well known to those
of ordinary skill in the pharmaceutical arts. The compounds can be
administered alone but generally will be administered with a
pharmaceutical carrier selected upon the basis of the chosen route
of administration and standard pharmaceutical practice. Compounds
of this invention can also be administered in intranasal form by
topical use of suitable intranasal vehicles, or by transdermal
routes, using transdermal skin patches. When compounds of this
invention are administered transdermally the dosage will be
continuous throughout the dosage regimen.
[0043] One aspect of the present invention involves administration
of the compounds of the present invention, or pharmaceutically
acceptable salts or solvates thereof, to a mammal implanted with a
tumor or susceptible to cancer formation. In general the compound
would be given in a dose range of from about 0.01 mg/kg to about
the MTD (maximum tolerated dose). The dosage and dosage regimen and
scheduling of a compound of the present invention must in each case
be carefully adjusted, utilizing sound professional judgment and
considering the age, weight and condition of the recipient, the
route of administration and the nature or extent of the cancer
disease condition. The term "systemic administration" as used
herein refers to oral sublingual, buccal, transnasal, transdermal,
rectal, intramascular, intravenous, intraventricular, intrathecal,
and subcutaneous routes. In accordance with good clinical practice,
it is preferred to administer the instant compounds at a
concentration level which will produce effective beneficial effects
without causing any harmful or untoward side effects.
DETAILED DESCRIPTION
[0044] Procedures for the preparation of Formula (I) compounds are
illustrated in Schemes 1-6 and the preparation of the key
intermediates/starting materials is illustrated in Scheme 7. 3 4 5
6 7 8 9
[0045] In Scheme 1, a selectively protected glycoside (1) was
treated under Mitsunobu conditions (cf. O. Mitsunobu Synth., 1981,
1, 1), for example using triphenylphosphine and diisopropyl
azodicarboxylate (DIAD), in a suitable solvent like benzene at room
temperature to 100.degree. C., preferably at or around 80.degree.
C., to give a 4'-bridged glycoside (2). Removal of the benzyl
protecting groups could then be done using a conventional procedure
involving hydrogenolysis over Pearlman's catalyst (20% Pd(OH).sub.2
on charcoal) to give a fully deprotected bridged glycoside (3).
Alternatively, a partially deprotected glycoside could be prepared
by treatment of the corresponding perbenzylated glycoside with
iodine in acetic anhydride (cf. K. P. R. Kartha, R. A. Field
Tetrahedron 1997, 53, 11753), followed by hydrolysis of the
intermediate acetate. Subsequent treatment of this selectively
deprotected glycoside with the well-known fluorinating agent DAST
[(diethylamino)sulfur trifluoride], followed by debenzylation as
before, then gives a monofluorinated bridged glycoside (4).
[0046] An alternative bridging procedure is shown in Scheme 2.
Deprotection of the imide moiety of perbenzylated glycosides such
as 5 was done by base-induced hydrolysis, followed by acidification
to give an intermediate anhydride. The latter was conveniently
converted to an imide using a suitable amine, such as that provided
by reaction with a mixture of hexamethyldisilazane and methanol in
dimethylformamide (cf. P. D Davis, R. A. Bit Tetrahedron Lett.
1990, 31, 5201). Selectively deprotected glycosides like 7 could
then be prepared by treatment of the corresponding perbenzylated
glycosides with iodine in acetic anhydride (cf. K. P. R. Kartha, R.
A. Field Tetrahedron 1997, 53, 11753), followed by hydrolysis of
the intermediate acetates. The resulting primary alcohol could then
be activated, for example as its mesylate and subsequently the
corresponding iodide, and induced to undergo elimination of the
element of HI using a suitable amine base, such as
1,8-diazabicyclo[5.4.0]undec-7- -ene (DBU), to give a vinyl ether
8. Treatment of this vinyl ether with iodine, in the presence of a
suitable base such as potassium tert-butoxide, leads to a bridging
reaction to give a 1', 5'-bridged glycoside 9. The resulting iodide
(9) readily undergoes a radical-induced reduction, using for
instance tri-n-butyltin hydride as the hydride source and 2,
2'-azobisisobutyronitrile (AIBN) as a radical initiator, to give
the corresponding 6'-deoxy bridged glycoside. Final removal of the
benzyl protecting groups to give 10 could be effected using a
number of standard methods, for example by treatment with boron
tribromide. Alternatively, the iodide 9 could also be readily
oxidized using a peracid, such as m-chloroperbenzoic acid, to give
a 6'-hydroxy-substituted bridged glycoside (11). Final deprotection
as before then gives derivative 12.
[0047] The aromatic core is also readily reduced as shown in Scheme
3. Following radical reduction of the iodide 13, the imide moiety
is reduced by treatment with a reducing hydride, such as sodium
borohydride, with further reduction using benzeneselenol to give a
mixture of the corresponding lactams. Final deprotection as before
then gives a mixture of the regioisomeric lactams 15 and 16.
[0048] In some instances, unprotected glycosides may conveniently
be used to prepare bridged glycosides. For instance, as shown in
Scheme 4, an unprotected glycoside (17) cyclizes under the
previously mentioned Mitsunobu reaction conditions to give a 1',
6'-bridged glycoside (18). This strategy is advantageous in that it
obviates the need for a final deprotection step.
[0049] Another useful approach to the synthesis 1', 6'-bridged
glycosides is shown in Scheme 5. In this case, the previously
mentioned glycoside (5) is mono-debenzylated as descibed before to
give the 6'-deprotected glycoside 19. The latter undergoes a
bridging reaction to give 20 under Mitsunobu conditions, for
example by the use of a complex of trimethylphosphine and
1,1'-(azodicarbonyl)dipiperidine (ADDP) in a suitable solvent, such
as benzene, at room temperature to around 100.degree. C.,
preferably at or about 80.degree. C. Removal of all of the benzyl
protecting groups is then done as before to give the 1',6'-bridged
glycoside 21.
[0050] Mono-debenzylated glycosides may also be used to prepare
selected bridged glycosides as shown in Scheme 6. Thus, the
unprotected hydroxyl group of the tri-O-benzylglycoside 22 could be
activated, for example as its mesylate, which may then undergo
elimination of the element of methanesulfonic acid using a suitable
amine base, such as diisopropylethylamine, to give a 1',2'-bridged
glycoside which is readily debenzylated as before to give 23.
[0051] A key intermediate sugar was prepared as shown in Scheme 7.
Conversion of a commercially available
methyl-.alpha.-D-glucopyranoside (24) to a 4-deoxyglycoside (26)
was done as reported by Barrette and Goodman (J. Org. Chem. 1984,
49, 176). Deprotection of the anomeric position could be done in
two steps, first by treatment with benzenethiol and a Lewis acid,
such as boron trifluoride etherate (cf. L. A. Paquette, J. A.
Oplinger J. Org. Chem. 1988, 53, 2953), followed by hydrolysis of
the resulting phenylthio sugar derivative (27) using
N-bromosuccinimide in a suitable solvent, such as acetone or
acetonitrile, in the presence of water (cf. B. Fraser-Reid, et al.
J. Am. Chem. Soc. 1988, 110, 2662). Alternatively, deprotection of
the anomeric position could be effected in one step by treatment
with a suitable acid, such as 90% formic acid, to give the
glucopyranoside (28) directly. Conversion of a glycopyranoside,
such as 28, to a glycopyranosyl chloride (29) could be done
according to a procedure reported by Iversen and Bundle (Carb. Res.
1982, 103, 29).
[0052] The compounds which constitute this invention and their
methods of preparation will appear more fully from a consideration
of the following examples which are given for the purpose of
illustration only and are not to be construed as in any way
limiting the scope of the invention.
Synthesis of Intermediates
[0053] Several intermediate compounds as well as other conventional
starting materials, used in the preparation of final products of
Formula I, were generally known in the literature or were
commercially available. Representative syntheses of some of these
compounds are nevertheless provided hereinbelow.
[0054] All anhydrous reactions were performed under an atmosphere
of nitrogen or argon using either commercially available dry
solvents or freshly distilled solvents. Melting points were
determined in an open capillary tube with a Thomas-Hoover melting
point apparatus and are uncorrected. Column chromatography was
performed using EM Science silica gel 60 (230-400 mesh) with the
designated solvent system as eluant. Thin-layer chromatography was
done on E. Merck silica gel 60 F.sub.254 plates (0.5 mm). Hplc
purity determinations were done using either a Shimadzu LC-10AS
with a SPD-10AV UV-Vis detector and one of the following columns;
YMC Combiscreen ODS-A (4.6.times.50 mm), or HP Zorbax SB-C18
(4.6.times.750 mm); or, an HP 1090 DR5 with a diode array detector
and a Waters Nova-Pak C18 column (3.9.times.150 mm). Infrared
spectra were recorded on a Nicolet Protg 460 FTIR as thin films or
KBr pellets. .sup.1HNMR spectra were recorded on either a Bruker
AMX-400 or a Bruker ARX-500 NMR spectrometer and chemical shifts
are expressed in parts per million (ppm or .delta.) with the
solvent in use as internal standard. Coupling constants are given
in hertz (Hz) and multiplets are designated as follows; singlet
(s), doublet (d), triplet (t), quartet (q), muliplet (m), and broad
(br). Low resolution mass spectra were determined on a Finnigan
Matt TSQ-7000 triple stage quadrapole spectrometer
(positive/negative ESI) operated in the negative ion mode.
EXAMPLE 1
6-(4-tert-Butylbenzyl)-2,3,9,10-tetrafluoro-12,13-[1,6-(2,3,4-tri-O-benzyl-
-.beta.-D-glucopyranosyl)]-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,-
4-c]carbazole-5,7-dione
[0055] 10
[0056] To a solution of
6-(4-tert-butylbenzyl)-2,3,9,10-tetrafluoro-12-(2,-
3,4-tri-O-benzyl-.beta.-D-glucopyranosyl)-6,7,12,13-tetrahydro(5H)indolo[2-
,3-a]pyrrolo[3,4-c]carbazole-5,7-dione (0.377 g, 0.39 mmol) in 10
mL of dry benzene was added a solution of trimethylphosphine (1 M
in THF, 0.78 mL, 0.78 mmol), followed by solid
1,1'-(azodicarbonyl)dipiperidine (ADDP) (0.227 mg, 0.90 mmol).
After stirring at room temperature under Ar for 5 min, the
resulting blood-red mixture was heated to reflux for 30 min. The
cooled yellow-orange mixture was applied directly onto a silica gel
flash column. Elution with dichloromethane afforded the title
compound (0.276 g, 74%) as a bright yellow glass:
[0057] .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 9.07 (dd, J=10.5,
8.4 Hz, 1H), 8.99 (dd, J=10.5, 8.3 Hz, 1H), 7.7-7.3 (m, 15H), 6.82
(br s, 3H), 6.47 (br s, 2H), 6.27 (m, 1H), 5.99 (d, J=7.7 Hz, 1H),
5.00 (d, J=12.7 Hz, 1H), 4.99 (d, J=11.0 Hz, 1H), 4.96 (d, J=11.0
Hz, 1H), 4.82 (d, J=12.6 Hz, 1H), 4.81 (d, J=15.0 Hz, 1H), 4.73 (d,
J=14.7 Hz, 1H), 4.61 (m, 1H), 4.45 (br s, 1H), 4.18-4.11 (m, 2H),
3.97-3.92 (m, 4H), 1.26 (s, 9H).
EXAMPLE 2
2,3,9,10-Tetrafluoro-12,13-[1,6-(2,3,4-tri-O-benzyl-.beta.-D-glucopyranosy-
l)]-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione
[0058] 11
[0059] To a solution of
6-(4-tert-butylbenzyl)-2,3,9,10-tetrafluoro-12,13--
[1,6-(2,3,4-tri-O-benzyl-.beta.-D-glucopyranosyl)]-6,7,12,13-tetrahydro(5h-
)indolo[2,3-a]pyrrolo[3,4-c]-carbazole-5,7-dione (0.276 g, 0.29
mmol) in 20 mL of ethanol-THF (1:1) was added 5 mL of 5 M KOH
solution. The resulting mixture was heated to reflux under Ar for 2
h while the THF was gradually allowed to distill off (Dean-Stark
trap). The reaction mixture was cooled at 0.degree. C., acidified
with conc. HCl and then diluted with ethyl acetate. This mixture
was vigorously stirred at r.t. for 1 h and then the organic phase
was separated and the aqueous phase was re-extracted with ethyl
acetate. The combined organic phase was washed (brine), dried
(Na.sub.2SO.sub.4) and evaporated to give a solid. The solid was
taken up in dichloromethane-acetonitrile and the solution was
concentrated to give a precipitate. The solid was filtered, washed
with a little acetonitrile and dried in vacuo to give 195 mg (83%)
of the anhydride as a bright yellow solid. To a solution of this
anhydride in 10 mL of dry DMF was added methanol (0.105 mL, 10
equiv), followed by hexamethyldisilazane (1.09 mL, 20 equiv). After
being stirred at r.t. for 7 h, the mixture was partitioned with
ethyl acetate-saturated aqueous NH.sub.4Cl, the organic phase was
separated and the aqueous phase was re-extracted with ethyl
acetate. The combined organic phase was washed (brine), dried
(Na.sub.2SO.sub.4) and evaporated to give a solid. Flash
chromatography (SiO.sub.2/dichloromethane-ethyl acetate, 1:0 then
4:1) afforded the pure product (0.183 g, 78% overall) as a yellow
glass:
[0060] .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 8.87 (m, 1H), 8.74
(m, 1H), 7.8-7.3 (m, 12H), 7.12 (br, 1H), 7.00 (br, 2H), 6.65 (br,
1H), 6.30 (br, 1H), 5.98 (d, J=7.4 Hz, 1H), 4.99 (m, 3H), 4.80 (d,
J=12.5 Hz, 1H), 4.54 (br s, 2H), 4.25-3.85 (m, 6H). MS (ESI.sup.-)
m/e 810 (M-H).sup.-.
EXAMPLE 3
2,3,9,10-Tetrafluoro-12,13-(1,6-.beta.-D-glucopyranosyl)-6,7,12,13-tetrahy-
dro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione
[0061] 12
[0062] To a solution of
2,3,9,10-tetrafluoro-12,13-[1,6-(2,3,4-tri-O-benzy-
l-.beta.-D-glucopyranosyl)]-6,7,12,13-tetrahydro(5R)indolo[2,3-a]pyrrolo[3-
,4-c]carbazole-5,7-dione (0.086 g, 0.106 mmol) in 40 mL of
chloroform-methanol (1:1) was added 20% Pd(OH).sub.2/C (0.090 g)
and the mixture was hydrogenated (balloon pressure) at room
temperature with vigorous stirring for 72 h. The resulting mixture
was then filtered (Celite) and the cake was washed successively
with methanol, THF and again with methanol. Evaporation of the
filtrate gave a brownish-yellow gum which was taken up in a little
methanol to give a solid. The solid was filtered, washed with a
little methanol and dried in vacuo to give the title compound as a
yellow solid. The filtrate was evaporated and the residue again
taken up in a minimum volume of methanol and allowed to stand at
room temperature to give additional pure product. Total yield was
0.038 g (60%): IR (KBr) 1749, 1700, 1487, 1473 cm.sup.-1.
[0063] .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta. 11.17 (s, 1H),
8.99 (dd, J=11.0, 9.1 Hz, 1H), 8.89 (dd, J=10.8, 8.6 Hz, 1H), 8.37
(dd, J=12.6, 7.2 Hz, 1H), 8.10 (dd, J=11.6, 6.8 Hz, 1H), 6.17 (br
s, 1H), 5.95 (d, J=7.9 Hz, 1H), 5.57 (d, J=4.6 Hz, 1H), 5.53 (d,
J=4.6 Hz, 1H), 4.89 (m, 1H), 4.57 (m, 1H), 4.41 (br m, 1H), 4.30
(dd, J=12.2, 2.9 Hz, 1H), 3.76 (d, J=5.7 Hz, 1H), 3.68 (m, 1H).
[0064] MS (ESI.sup.-) m/e 540 (M-H).sup.-.
[0065] HPLC: 97.2% (320 nm).
EXAMPLE 4
2,3,9,10-Tetrafluoro-12,13-[1,6-(4-deoxy-.beta.-D-glucopyranosyl)]-6,7,12,-
13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione
[0066] 13
[0067] Prepared as per Example 3 as a yellow solid in 69%
yield:
[0068] IR (KBr) 1717, 1700, 1487, 1473 cm.sup.-1.
[0069] .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta. 9.19 (m, 1H),
9.09 (m, 1H), 8.37 (dd, J=12.5, 7.1 Hz, 1H), 7.69 (dd, J=11.2, 6.6
Hz, 1H), 5.90 (d, J=7.5 Hz, 1H), 4.64 (m, 3H), 4.48 (br s, 1H),
3.96 (dt, J=9.6, 8.6 Hz, 1H), 2.84 (dt, J=13.4, 8.5, 1H).
[0070] MS (ESI.sup.-) m/e 524 (M-H).sup.-.
[0071] HPLC: 99.6% (320 nm).
EXAMPLE 5
2,3,9,10-Tetrafluoro-12,13-[1,6-(3-deoxy-.beta.-D-glucopyranosyl)]-6,7,12,-
13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione
[0072] 14
[0073] Prepared as per Example 3 as a yellow solid in 75%
yield:
[0074] .sup.1H NMR (acetone-d.sub.6, 400 MHz) .delta. 9.35 (s, 1H),
8.63 (m, 1H), 8.56 (m, 1H), 7.95 (m, 1H), 7.51 (m, 1H), 5.52 (d,
J=5.6 Hz, 1H), 5.20 (br s, 1H), 4.45 (m, 3H), 4.18 (d, J=12.7 Hz,
1H), 4.07 (m, 1H), 3.91 (m, 1H), 2.50 (m, 1H), 1.84 (m, 1H).
[0075] MS (ESI.sup.-) m/e 524 (M-H).sup.-.
[0076] HPLC: 98.5% (320 nm).
EXAMPLE 6
2,3,9,10-Tetrafluoro-12,13-[1,6-(3-deoxy-3-fluoro-.beta.-D-glucopyranosyl)-
]-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione
[0077] 15
[0078] Prepared as per Example 3 as a yellow solid in 58%
yield:
[0079] IR (KBr) 1826, 1757, 1489, 1475 cm.sup.-1.
[0080] .sup.1H NMR (THF-d.sub.8, 400 MHz) .delta. 10.83 (s, 1H),
8.92-8.80 (m, 2H), 8.48 (m, 1H), 7.88 (m, 1H), 6.38 (br s, 1H),
6.16 (d, J=7.8 Hz, 1H), 5.47 (br s, 1H), 4.96 (m, 2H), 4.84-4.67
(m, 2H), 4.56 (d, J=12.5 Hz, 1H), 4.15 (dt, J=25.0, 5.5 Hz,
1H).
[0081] MS (ESI.sup.-) m/e 542 (M-H).sup.-.
[0082] HPLC: 98.5% (320 nm).
EXAMPLE 7
1,11-Dichloro-12,13-[1,6-(4-O-methyl-.beta.-D-glucopyranosyl)]-6,7,12,13-t-
etrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione
[0083] 16
[0084] To a suspension of
1,11-dichloro-12-(4-O-methyl-.beta.-D-glucopyran-
osyl)]-6,7,12,13-tetrahydro(5H1)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-d-
ione (0.184 g, 0.32 mmol) in 10 mL of THF, at room temperature
under Ar, was added triphenylphosphine (0.262 g, 1.0 mmol) followed
by diisopropyl azodicarboxylate (DIAD) (0.193 mL, 1.0 mmol). The
resulting mixture was stirred at room temperature for 3 days and
then it was filtered and the filtrate was evaporated to give a
yellow gum. This gum was triturated with dichloromethane and the
mixture was filtered to give a solid which was taken up in methanol
and vigorously stirred, whereupon a solid separated. The solid was
isolated by filtration and dried in vacuo to give the title
compound (0.113 g, 64%) as a bright yellow solid:
[0085] IR(KBr) 1711, 1321, 1064 cm.sup.-1.
[0086] .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta. 11.44 (s, 1H),
9.26 (d, J=7.9 Hz, 1H), 9.15 (d, J=7.9 Hz, 1H), 7.76 (d, J=7.4 Hz,
1H), 7.61 (d, J=7.8 Hz, 1H), 7.50 (t, J=7.9 Hz, 1H), 7.41 (t, J=7.9
Hz, 1H), 6.61 (d, J=7.3 Hz, 1H), 5.70 (dd, J=16.8, 5.8 Hz, 1H),
5.15 (d, J=5.9 Hz, 1H), 4.91 (d, J=6.0 Hz, 1H), 4.80 (d, J=5.6 Hz,
1H), 4.23 (d, J=5.0 Hz, 1H), 4.21 (d, J=16.1 Hz, 1H), 3.58 (s, 3H),
3.54 (m, 1H), 3.44 (m, 1H).
[0087] MS (ESI.sup.-) m/e 550 (M-H).sup.-.
[0088] HPLC: 97.3% (320 nm).
EXAMPLE 8
2,3,9,10-Tetrafluoro-12,13-[1,6-(4-deoxy-4-fluoro-.beta.-D-glucopyranosyl)-
]-6,7,12,13-tetrahydro(5H)indolo[2,3-alpyrrolo[3,4-c]carbazole-5,7-dione
[0089] 17
[0090] Prepared as per Example 7 as a yellow solid in 48%
yield:
[0091] IR (KBr) 1750, 1700, 1488, 1473 cm.sup.-1.
[0092] .sup.1H NMR (THF-d.sub.8, 400 MHz) .delta. 10.06 (s, 1H),
9.17 (dd, J=11.3, 8.6 Hz, 1H), 9.07 (dd, J=11.1, 8.4 Hz, 1H), 8.36
(dd, J=12.3, 6.9 Hz, 1H), 7.79 (dd, J=11.3, 6.6 Hz, 1H), 6.04 (d,
J=7.8 Hz, 1H), 5.93 (br s, 1H), 5.61 (br s, 1H), 4.87-4.50 (m, 5H),
4.10 (m, 1H).
[0093] MS (ESI.sup.-) m/e 542 (M-H).sup.-.
[0094] HPLC: 99.0% (320 nm).
EXAMPLE 9
2,3,9,10-Tetrafluoro-12,13-[1,6-(4-deoxy-4,4-difluoro-.beta.-D-glucopyrano-
syl)]-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dio-
ne
[0095] 18
[0096] Prepared as per Example 7 as a yellow solid in 6% yield:
[0097] .sup.1H NMR (acetone-d.sub.6, 400 MHz) .delta. 9.85 (s, 1H),
8.96 (dd, J=11.3, 8.7 Hz, 1H), 8.90 (dd, J=11.0, 8.5 Hz, 1H), 8.29
(dd, J=12.4, 7.1 Hz, 1H), 7.99 (dd, J=11.5, 6.7 Hz, 1H), 6.09 (d,
J=8.0 Hz, 1H), 4.98-4.70 (m, 4H), 4.31 (dt, J=15.4, 11.3 Hz,
1H).
[0098] MS (ESI.sup.-) m/e 560 (M-H).sup.-.
[0099] HPLC: 95.2% (320 nm).
EXAMPLE 10
2,10-Difluoro-12,13-[1,6-.beta.-D-glucopyranosyl]-6,7,12,13-tetrahydro(5M)-
indolo-[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione
[0100] 19
[0101] Prepared as per Example 7 in 12% yield as a yellow solid, mp
>300.degree. C.:
[0102] IR(KBr)3414(br), 1744, 1702, 1619, 1582, 1490, 1468, 1449,
1415, 1328, 1182, 1112, 1043, 826, 763 cm.sup.-1.
[0103] .sup.1H NMR (DMSO-d.sub.6, 500 MHz) .delta. 9.22 (dd, J=8.6,
6.3 Hz, 1H), 9.14 (dd, J=8.6, 6.0 Hz, 1H), 8.14 (d, J=9.9 Hz, 1H),
7.81 (dd, J=10.5, 1.8 Hz, 1H), 7.24-7.18 (m, 2H), 5.98 (d, J=7.7
Hz, 1H), 5.63 (br s, 1.5H), 4.85-4.83 (m, 1H), 4.65-4.60 (m, 1H),
4.49 (m, 1H), 4.35-4.32 (m, 1H), 3.80 (d, J=6.3 Hz, 1H), 3.68 (dd,
J=10.8, 6.3 Hz, 1H). MS (ESI.sup.-) m/e 504 (M-H).sup.-.
EXAMPLE 11
2,10-Difluoro-12,13-[1,6-(4-deoxy-4-fluoro-.beta.-D-glucopyranosyl)]-6,7,1-
2,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione
[0104] 20
[0105] Prepared as per Example 7 and isolated in 24% yield after
LH-20 chromatography (MeOH) as a yellow solid, mp 315.degree. C.
(dec):
[0106] IR (KBr) 3423, 2926, 1747, 1702, 1619, 1583, 1490, 1467,
1449, 1415, 1358, 1222, 1182, 1146, 1110, 1040, 996, 937, 827, 763,
641 cm.sup.-1.
[0107] .sup.1H NMR (DMSO-d.sub.6, 500 MHz) .delta. 9.21-9.19 (m,
1H), 9.12-9.10 (m, 1H), 8.10 (d, J=10.9 Hz, 1H), 7.92 (d, J=10.4
Hz, 1H), 7.21-7.15 (m, 2H), 6.08 (d, J=7.6 Hz, 1H), 5.04-5.01 (m,
1H), 4.87 (dd, JHF=50.7, 5.3 Hz, 1H), 4.69-4.60 (2m, 2H), 4.55 (m,
1H), 3.98 (ddd, J.sub.HF=28.0, J=10.8, 5.4 Hz, 1H).
[0108] MS (ESI.sup.-) m/e 506 (M-H).sup.-.
EXAMPLE 12
2,3,9,10-Tetrafluoro-12,13-[1,4-(2,3,6-tri-O-benzyl-.beta.-D-galactopyrano-
syl)]-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dio-
ne
[0109] 21
[0110] To a solution of
2,3,9,10-tetrafluoro-12-(2,3,6-tri-O-benzyl-.beta.-
-D-glucopyranosyl)-
6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]car-
bazole-5,7-dione (0.168 g, 0.20 mmol) and triphenylphosphine (0.157
g, 0.60 mmol) in 5 mL of dry benzene was added diisopropyl
azodicarboxylate (DIAD) (0.118 mL, 0.60 mmol) dropwise at room
temperature under Ar. After stirring at room temperature for 15
min, the resulting blood-red mixture was heated to reflux for 30
min. The cooled mixture was evaporated and the resulting orange gum
was chromatographed (SiO.sub.2/dichloromethane-a- cetonitrile, 1:0
then 97:3) to give the title compound (0.124 g, 77%) as a bright
yellow glass:
[0111] .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 9.17 (dd, J=10.7,
8.3, 1H), 9.00 (dd, J=10.6, 8.2 Hz, 1H), 7.65 (br s, 1H), 7.48-7.30
(m, 9H), 7.20 (m, 3H), 7.13 (dd, J=7.6, 7.2 Hz, 2H), 6.67 (d, J=7.3
Hz, 2H), 6.61 (m, 1H), 6.33 (s, 1H), 5.52 (dd, J=8.5, 2.0 Hz, 1H),
4.84 (d, J=12.2 Hz, 1H), 4.83 (m, 1H), 4.54 (d, J=12.2 Hz, 1H),
4.34 (d, J=8.4 Hz, 1H), 4.31 (d, J=11.2 Hz, 1H), 4.04 (m, 2H), 3.95
(d, J=1.4 Hz, 1H), 3.56 (dd, J=8.9, 4.7 Hz, 1H), 3.02 (t, J=9.2 Hz,
1H).
[0112] MS (ESI.sup.-) m/e 810 (M-H).sup.-.
EXAMPLE 13
2,3,9,10-Tetrafluoro-12,13-(1,4-.beta.-D-galactopyranosyl]-6,7,12,13-tetra-
hydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione
[0113] 22
[0114] A mixture of
2,3,9,10-tetrafluoro-12,13-[1,4-(2,3,6-tri-O-benzyl-.b-
eta.-D-galactopyranosyl)-
6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-
-c]carbazole-5,7-dione (0.059 g, 0.073 mmol) and 20% Pd(OH).sub.2/C
(0.059 g) in 10 mL of chloroform-methanol (1:1) was hydrogenated
(balloon pressure) at room temperature with vigorous stirring for
17 h. The resulting mixture was then filtered (Celite) and the cake
was washed successively with THF, methanol and dichloromethane.
Evaporation of the filtrate gave a solid which was taken up in THF
and filtered. The filtrate was evaporated and the residue was
triturated with a minimum volume of cold methanol, filtered and
dried in vacuo. This afforded the title compound (0.032 g, 80%) as
a bright yellow solid:
[0115] IR (KBr) 1750, 1700, 1487, 1473 cm .sup.1.
[0116] .sup.1H NMR (THF-d.sub.8, 400 MHz) .delta. 10.13 (br s, 1H),
9.20 (m, 2H), 7.79 (dd, J=12.0, 6.6 Hz, 1H), 7.71 (dd, J=11.4, 6.5
Hz, 1H), 6.56 (s, 1H), 5.71 (dd, J=7.9,2.8 Hz, 1H), 5.46 (d, J=4.0
Hz, 1H), 4.76 (m, 1H), 4.63 (d, J=4.5 Hz, 1H), 4.46 (m, 1H), 4.12
(dd, J=5.6, 3.6 Hz, 1H), 4.05 (m, 1H), 3.43 (m, 1H), 2.97 (m,
1H).
[0117] MS (ESI.sup.-) m/e 540 (M-H).sup.-.
[0118] HPLC: 95.5% (320 nm).
EXAMPLE 14
2,3,9,10-Tetrafluoro-12,13-[1,4-(2,3-di-O-benzyl-.beta.-D-galactopyranosyl-
)]-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione
[0119] 23
[0120] A mixture of
2,3,9,10-tetrafluoro-12,13-[1,4-(2,3-di-O-benzyl-.beta-
.-D-galactopyranosyl)]-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]-
carbazole-5,7-dione (0.124 g, 0.15 mmol) and iodine (0.010 mg,
0.038 mmol) in 5 mL of acetic anhydride was stirred at
room'temperature under Ar for 16 h. The resulting mixture was then
evaporated in vacuo and the residue was taken up in toluene and
again evaporated to give a yellow gum. This material was then taken
up in 15 mL of methanol, 3 mL of ammonium hydroxide solution was
added, the flask was sealed and the mixture was stirred at room
temperature for 20 h. The mixture was then evaporated in vacuo and
the residue was taken up in toluene and again evaporated to give a
yellow gum. Flash chromatography of this material
(SiO.sub.2/dichloromethane-ethyl acetate, 9:1) afforded the title
compound (0.078 g, 72%) as a yellow glass:
[0121] .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 8.81 (m, 1H), 8.65
(m, 1H), 7.85 (s, 1H), 7.38 (m, 6H), 7.22-7.14 (m, 3H), 6.78 (d,
J=6.9 Hz, 2H), 6.54 (dd, J=10.4, 6.3 Hz, 1H), 6.29 (s, 1H), 5.47
(d, J=7.9 Hz, 1H), 4.81 (d, J=12.2 Hz, 1H), 4.75 (m, 1H), 4.52 (d,
J=12.3 Hz, 1H), 4.36 (d, J=7.7 Hz, 1H), 4.15 (m, 2H), 3.96 (s, 1H),
3.67 (dd, J=10.8, 5.5 Hz, 1H), 3.39 (dd, J=10.6, 8.1 Hz, 1H), 2.06
(br s, 1H).
[0122] MS (ESI.sup.-) m/e 720 (M-H).sup.-.
EXAMPLE 15
2,3,9,10-Tetrafluoro-12,13-[1,4-(6-deoxy-6-fluoro-2,3-di-O-benzyl-.beta.-D-
-galactopyranosyl)]-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]car-
bazole-5,7-dione
[0123] 24
[0124] To a solution of
2,3,9,10-tetrafluoro-12,13-[1,4-(2,3-di-O-benzyl-.-
beta.-D-galactopyranosyl)-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-
-c]carbazole-5,7-dione (0.232 g, 0.32 mmol) in 7.5 mL of dry
dichloromethane was added (diethylamino)sulfur trifluoride (DAST)
(0.127 mL, 0.96 mmol) dropwise at -40.degree. C. under Ar. After 20
min the cooling bath was removed and the mixture was stirred at
room temperature for 3 h. The reaction mixture was then recooled at
40.degree. C. and DAST (0.065 mL, 0.49 mmol) was again added. The
mixture was then stirred at room temperature for 3 h, recooled at
-40.degree. C., quenched with methanol (10 mL) and evaporated to
give a gum. Flash chromatography (SiO.sub.2/dichloromethane-ethyl
acetate, 1:0 then 95:5) of this material afforded the title
compound (0.168 g, 72%) as a bright yellow glass:
[0125] .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 9.13 (dd, J=10.7,
8.3 Hz, 1H), 8.93 (dd, J=10.7, 8.2 Hz, 1H), 7.71 (s, 1H), 7.48 (m,
6H), 7.33 (dd, J=10.7, 6.2 Hz, 1H), 7.22 (t, J=7.3 Hz, 1H), 7.16
(dd, J=7.6, 7.1 Hz, 2H), 6.68 (d, J=7.1 Hz, 2H), 6.54 (dd, J=10.5,
6.3 Hz, 1H), 6.33 (s, 1H), 5.45 (d, J=8.6 Hz, 1H), 4.94 (m, 1H),
4.89 (d, J=12.2 Hz, 1H), 4.58 (d, J=12.3 Hz, 1H), 4.50 (dd, J=9.2,
5.2 Hz, 0.5H), 4.39 (d, J=8.2 Hz, 1H), 4.38 (m, 0.5H), 4.05 (m,
1H), 3.98 (m, 1H).
[0126] MS (ESI.sup.-) m/e 722 (M-H).sup.-.
EXAMPLE 16
2,3,9,10-Tetrafluoro-12,13-[1,4-(6-deoxy-6-fluoro-.beta.-D-galactopyranosy-
l)]-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione
[0127] 25
[0128] A mixture of
2,3,9,10-tetrafluoro-12,13-[1,4-(2,3-di-O-benzyl-6-deo-
xy-6-fluoro-.beta.-D-galactopyranosyl)-6,7,12,13-tetrahydro(5H)indolo[2,3--
a]pyrrolo[3,4-c]carbazole-5,7-dione (0.168 g, 0.23 mmol) and 20%
Pd(OH).sub.2/C (0.185 g) in 25 mL of chloroform-methanol (1: 1) was
hydrogenated (balloon pressure) at r.t. with vigorous stirring for
3 days. The resulting mixture was then filtered (Celite) and the
cake was washed with methanol and then THF, and the filtrate was
evaporated to give a solid. This material was chromatographed
(SiO.sub.2/dichloromethan- e-methanol-THF, 8:1:1) and the
product-containing fractions were combined and rechromatographed
(SiO.sub.2/dichloromethane-methanol-THF, 90:5:5) to give the pure
title compound (0.096 g, 77%) as a bright yellow solid:
[0129] IR (KBr) 17530, 1716, 1487, 1474 cm.sup.-1.
[0130] .sup.1H NMR (acetone-d.sub.6, 400 MHz) .delta. 9.22 (dd,
J=11.2, 8.5 Hz, 1H), 9.18 (dd, J=11.3, 8.4 Hz, 1H), 7.90 (dd,
J=11.7, 6.6 Hz, 1H), 7.87 (dd, J=11.8, 6.5 Hz, 1H), 6.77 (s, 1H),
5.96 (dd, J=8.6, 2.8 Hz, 1H), 5.10 (m, 1H), 4.70 (dd, J=6.5, 1.5
Hz, 1H), 4.40 (dd, J=10.0, 5.3 Hz, 0.5H), 4.30 (m, 1H), 4.19 (dd,
J=9.7, 6.4 Hz, 0.5H), 3.29 (s, 1H).
[0131] MS (ESI.sup.-) m/e 542 (M-H).sup.-.
[0132] HPLC: 97.5% (320 nm).
EXAMPLE 17
2,3,9,10-Tetrafluoro-12-(2,3-di-O-benzyl-4,
6-dideoxy-5,6-anhydro-.beta.-D-
-glucopyranosyl)-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbaz-
ole-5,7-dione
[0133] 26
[0134] A mixture of
2,3,9,10-tetrafluoro-12-(2,3-di-O-benzyl-4-deoxy-.beta-
.-D-glucopyranosyl)-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]car-
bazole-5,7-dione (2.00 g, 2.76 mmol) and freshly activated,
pulverized 4A molecular sieves (0.60 g) in 100 mL of
dichloromethane was cooled at 5.degree. C. under Ar and
triethylamine (0.77 mL, 5.52 mmol), DMAP (0.20 g, 1.64 mmol) and
methanesulfonyl chloride (0.32 mL, 4.14 mmol) were added
sequentially. The mixture was stirred at the same temperature for 2
h and then it was filtered and the filter-cake was washed with
ethyl acetate. The flitrate was diluted with ethyl acetate (200 mL)
and ether (50 mL) and then it was washed (H.sub.2O .times.2,
brine), dried (MgSO.sub.4) and evaporated to give a yellow glass.
This material was taken up in 100 mL of acetone, NaI ( ) was added
and the mixture was heated to reflux under Ar for 18 h. The cooled
mixture was then evaporated to dryness and the residue was taken up
in 10 mL of ethyl acetate, washed (H.sub.2O .times.2, brine) dried
(MgSO.sub.4) and evaporated. The resulting solid was
chromatographed (SiO.sub.2/2-32% ethyl acetate-hexane) to give
2,3,9,10-tetrafluoro-12-(2,3-di-O-benzyl-4--
deoxy-6-iodo-.beta.-D-glucopyranosyl)-6,7,12,13-tetrahydro(5H)indolo[2,3-a-
]pyrrolo[3,4-c]carbazole-5,7-dione (0.90 g, 39%) as an amorphous
yellow solid. To an ice-cold solution of this iodide (0.500 g, 0.60
mmol) in 20 mL of dry THF was added DBU (0.27 mL, 1.80 mmol) and
the solution was kept at 5.degree. C. for 2 h. The cooling bath was
then removed and stirring was continued at room temperature for 16
h. Another portion of DBU (0.27 mL, 1.80 mmol) was then added and
the reaction was allowed to continue for another 24 h. A further
portion of DBU (0.27 mL, 1.80 mmol) was added and stirring was
continued for an additional 24 h. The resulting mixture was diluted
with ethyl acetate and then it was washed (1 N HCl .times.2,
H.sub.2O .times.2, 1 M NaHCO.sub.3 .times.2, H.sub.2O, brine),
dried (MgSO.sub.4) and evaporated to give a gum. Flash
chromatography (SiO.sub.2/2-16% ethyl acetate-hexane) afforded the
title compound (0.297 g, 70%) as a yellow solid:
[0135] .sup.1H NMR (acetone-d.sub.6, 400 MHz) .delta. 9.11 (dd,
J=11.0, 8.5 Hz, 1H), 8.95 (dd, J=11.0, 8.5 Hz, 1H), 7.94 (m, 1H),
7.46 (m, 2H), 7.37 (m, 4H), 6.84 (t, J=7.3 Hz, 1H), 6.69 (m, 2H),
6.57 (d, J=8.5 Hz, 1H), 6.43 (br s, 2H), 4.92 (d, J=11.4 Hz, 1H),
4.74(d, J=11.4 Hz, 1H), 4.69 (s, 1H), 4.58 (s, 1H), 4.40 (d, J=11.8
Hz, 1H), 4.26 (d, J=5.5 Hz, 2H), 3.99 (d, J=11.8 Hz, 1H), 3.25 (m,
1H).
[0136] MS (ESI.sup.-) m/e 704 (M-H).sup.-.
EXAMPLE 18
2,3,9,10-Tetrafluoro-12-(2,3,4-tri-O-benzyl-6-deoxy-5,6-anhydro-.beta.-D-g-
lucopyranosyl)-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazol-
e-5,7-dione
[0137] 27
[0138] Prepared as per Example 17 as a yellow solid in 54% overall
yield:
[0139] .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 9.38 (s, 1H), 9.13
(t, J=9.3 Hz, 1H), 8.99 (t, J=9.3 Hz, 1H), 7.56-7.41 (m, 6H), 7.29
(m, 2H), 6.90 (t, J=7.1 Hz, 1H), 6.74 (t, J=7.5 Hz, 2H), 6.68 (d,
J=7.9 Hz, 1H), 6.43 (d, J=7.2 Hz, 3H), 5.06 (m, 2H), 4.84 (d,
J=11.6 Hz, 1H), 4.74 (d, J=1.3 Hz, 1H), 4.68 (d, J=10.1 Hz, 1H),
4.58-4.54 (m, 2H), 4.19 (m, 1H), 3.94 (d, J=11.4 Hz, 1H).
[0140] MS (ESI.sup.-) m/e 810 (M-H).sup.-.
EXAMPLE 19
2,3,9,10-Tetrafluoro-12,13-[1,5-(6-deoxy-6-iodo-2,3,4-tri-O-benzyl-.beta.--
D-glucopyranosyl]-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carba-
zole-5,7-dione
[0141] 28
[0142] To a solution of
2,3,9,10-tetrafluoro-12-(2,3,4-tri-O-benzyl-6-deox-
y-5,6-anhydro-.beta.-D-glucopyranosyl)-6,7,12,13-tetrahydro(5H)indolo[2,3--
a]pyrrolo[3,4-c]carbazole-5,7-dione (0.047 g, 0.058 mmol) in a
mixture of THF (7 mL) and methanol (1 mL) was added potassium
tert-butoxide (0.025 g, 0.22 mmol), followed by iodine (0.047 g,
0.19 mmol). The resulting brown mixture was stirred at room
temperature under Ar for 18 h and then it was diluted with ethyl
acetate. This mixture was then washed (aq. NaS.sub.2O.sub.3,
brine), dried (Na.sub.2SO.sub.4) and evaporated to give a brown
residue. Purification by prep tlc (hexane-ethyl acetate, 2:1)
afforded the title compound (0.022 g, 41%) as a yellow solid:
[0143] .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 9.01 (dd, J=10.4
Hz, 8.6 Hz, 1H), 8.82 (dd, J=10.5 Hz, 8.2 Hz, 1H), 7.70 (s, 1H),
7.49 (m, 3H), 7.39 (d, J=3.6 Hz, 2H), 7.22-7.05 (m, 6H), 6.90-6.84
(m, 3H), 6.78 (d, J=7.3 Hz, 2H), 6.33 (dd, J=10.6 Hz, 6.2 Hz, 1H),
6.22 (d, J=4.9 Hz, 1H), 5.05 (d, J=11.9 Hz, 1H), 4.69 (t, J=l 1.1
Hz, 2H), 4.57-4.53 (m, 2H), 4.41 (d, J=11.1 Hz, 1H), 4.21 (d,
J=11.6 Hz, 1H), 4.16-4.11 (m, 1H), 4.08 (m, 3H).
[0144] MS (ESI.sup.-) m/e 936 (M-H).sup.-.
EXAMPLE 20
2,3,9,10-Tetrafluoro-12,13-[1,5-(4,6-dideoxy-6-iodo-2,3,4-tri-O-benzyl-.be-
ta.-D-glucopyranosyl)]-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]-
carbazole-5,7-dione
[0145] 29
[0146] Prepared as per Example 19 in 68% yield.
[0147] .sup.1H NMR (acetone-d.sub.6, 500 MHz) .delta. 9.10 (dd,
J=10.4 Hz, 8.6 Hz, 1H), 8.95 (dd, J=10.4 Hz, 8.6 Hz, 1H), 8.02 (m,
1H), 7.6-7.4 (m, 7H), 7.08 (m, 1H), 6.90 (m, 1H), 6.76-6.65 (m,
2H), 6.70 (d, J=1 Hz, 1H), 5.05 (d, J=11.5 Hz, 1H), 4.98 (d, J=11.5
Hz, 1H), 4.91 (d, J=12.3 Hz, 1H), 4.23 (dd, J=3.3, 3.3 Hz, 1H),
4.22 (d, J=12.3 Hz, 1H), 4.15 (dd, J=1, 3.3 Hz, 1H), 3.80 (d,
J=11.5 Hz, 1H), 3.73 (d, J=11.5 Hz, 1H), 3.25 (dd, J=3.3, 14.9 Hz,
1H), 3.01 (dd, J=3.3, 14.9 Hz, 1H).
[0148] MS (ESI.sup.-) m/e 830 (M-H).sup.-.
EXAMPLE 21
2,3,9,10-Tetrafluoro-12,13-1,5-(6-deoxy-.beta.-D-glucopyranosyl)]-6,7,12,1-
3-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione
[0149] 30
[0150] To a solution of
2,3,9,10-tetrafluoro-12,13-[1,5-(6-deoxy-6-iodo-2,-
3,4-tri-O-benzyl-.beta.-D-glucopyranosyl)]-6,7,12,13-tetrahydro(5H)indolo[-
2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione (0.025 g, 0.027 mmol) in 5
mL of dichloromethane, at -78.degree. C. under Ar, was added a
solution of boron tribromide (1 M in dichloromethane, 0.27 mL, 0.27
mmol) dropwise. After 30 min an additional 0.15 mL (0.15 mmol) of
boron tribromide solution was added and the reaction mixture was
allowed to warm to 0.degree. C. over 1 h. The mixture was then
recooled at -78.degree. C., 0.20 mL (0.20 mmol) of boron tribromide
solution was again added, and the mixture was allowed to warm to
0.degree. C. over 2 h. The reaction mixture was then quenched with
methanol, diluted with ethyl acetate, washed (brine), dried
(Na.sub.2SO.sub.4) and evaporated. The residue was purified by prep
tlc (THF-hexane, 2:1) to give the title compound (0.003 g, 21%) as
a yellow solid:
[0151] .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta. 11.26 (s, 1H),
8.97 (t, J=9.5 Hz, 1H), 8.76 (t, J=9.1 Hz, 1H), 8.01-7.91 (m, 2H),
6.46 (d, J=5.2 Hz, 1H), 6.26 (d, J=5.3 Hz, 1H), 5.87 (d, J=5.3 Hz,
1H), 5.38 (s, 2H), 4.21 (m, 1H), 4.10 (m, 1H), 3.79-3.71 (m, 2H),
3.16 (d, J=5.3 Hz, 1H).
[0152] MS (ESI.sup.-) m/e 540 (M-H).sup.-.
[0153] HPLC: 97.0% (320 nm).
EXAMPLE 22
2,3,9,10-Tetrafluoro-12,13-(1,5-.beta.-D-glucopyranosyl)-6,7,12,13-tetrahy-
dro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione
[0154] 31
[0155] To a solution of
2,3,9,10-tetrafluoro-12,13-[1,5-(6-deoxy-6-iodo-2,-
3,4-tri-O-benzyl-.beta..beta.-D-glucopyranosyl)]-6,7,12,13-tetrahydro(5H)i-
ndolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione (0.025 g, 0.027 mmol)
in 4 mL of dichloromethane containing several drops of water, was
added m-chloroperbenzoic acid (0.018 g, 0.11 mmol) and the
resulting mixture was stirred at room temperature for 3 days. The
mixture was then partitioned with ethyl acetate and saturated
aqueous NaHSO.sub.3.and the organic phase was separated, washed
(saturated aqueous NaHCO.sub.3, brine), dried (NaSO.sub.4) and
evaporated to give a solid. Purification by prep tlc afforded
2,3,9,10-tetrafluoro-12,13-[1,5-(6-hydroxy-2,3,4-tri-
-O-benzyl-.beta.-D-glucopyranosyl)]-6,7,12,13-tetrahydro(5H)indolo[2,3-a]p-
yrrolo[3,4-c]carbazole-5,7-dione (0.006 g, 27%) as a yellow solid:
MS (ESI.sup.-) m/e 826 (M-H).sup.-. This material was taken up in
dichloromethane (5 mL), the solution was cooled at -78.degree. C.
under Ar and a solution of boron tribromide (1 M in
dichloromethane, 0.073 mL, 0.073 mmol) was then added dropwise. The
resulting mixture was kept at -78.degree. C. for 15 min, then at
0.degree. C. for 75 min and finally it was quenched with methanol
and diluted with ethyl acetate. This mixture was then washed
(brine), dried (Na.sub.2SO.sub.4) and evaporated, and the residue
was purified by prep tlc (THF-hexane, 3:2) to give the title
compound (0.0016 g, 40%) as a yellow solid:
[0156] .sup.1H NMR (acetone-d.sub.6, 400 MHz) .delta. 10.01 (s,
1H), 9.09 (dd, J=11.4 Hz, 8.7 Hz, 1H), 8.88 (t, J=8.8 Hz, 1H),
7.88-7.76 (m, 2H), 6.55 (d, J=6.0 Hz, 1H), 4.79-4.67 (m, 2H),
4.26-4.18 (m, 2H), 4.12-4.06 (m, 1H), 3.89-3.78 (m, 2H), 3.52 (m,
1H), 3.29 (s, 1H).
[0157] MS (ESI.sup.-) m/e 556 (M-H).sup.-.
[0158] HPLC: 98.1% (320 nm).
EXAMPLE 23
2,3,9,10-Tetrafluoro-12,13-[1,5-(6-deoxy-.beta.-D-glucopyranosyl)]-6,7,12,-
13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5-one and
2,3,9,10-Tetrafluoro-12,13-[1,5-(6-deoxy-.beta.-D-glucopyranosyl)]-6,7,12-
,13-tetrahydro(5H)-indolo[2,3-a]pyrrolo[3,4-c]carbazole-7-one
[0159] 32
[0160] To a solution of
2,3,9,10-tetrafluoro-12,13-[1,5-(6-deoxy-2,3,4-tri-
-O-benzyl-.beta.-D-glucopyranosyl)]-6,7,12,13-tetrahydro(5H)indolo[2,3-a]p-
yrrolo[3,4-c]carbazole-5,7-dione (0.060 g, 0.074 mmol) in 10 mL of
ethanol was added sodium borohydride (0.027 g, 0.71 mmol) in three
portions at 1 h intervals. One hour after the final addition,
another 0.035 g (0.92 mmol) of sodium borohydride was added and the
resulting mixture was stirred at room tmeperature for 3 days. The
mixture was then diluted with ethyl acetate, washed (sat.
NH.sub.4Cl, sat. NaHCO.sub.3, brine), dried (Na.sub.2SO.sub.4) and
evaporated to give a solid residue. This residue was immediately
taken up in dichloromethane (5 mL) and phenylselenol (0.055 mL,
0.52 mmol) was added, followed by p-toluenesulfonic acid
monohydrate (0.003 g, 0.015 mmol). After 2 h at room temperature,
the resulting mixture was evaporated and the residue was purified
by prep tlc (dichloromethane-methanol, 97:3) to give a 1:1 mixture
of 2,3,9,10-tetrafluoro-12,13-[l,
5-(6-deoxy-2,3,4-tri-O-benzyl-.beta.-D-glu-
copyranosyl)]-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-
-5-one and
2,3,9,10-tetrafluoro-12,13-[1,5-(6-deoxy-2,3,4-tri-O-benzyl-.be-
ta.-D-glucopyranosyl)]-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]-
carbazole-7-one (0.014 g, 24%) as a nearly colourless solid: MS
(ESI) m/e 796 (M-H).sup.-. This material was taken up in
dichloromethane (5 mL), cooled at -78.degree. C. and then treated
with boron tribromide (1 M in dichloromethane, 0.18 mL, 0.18 mmol).
The mixture was stirred at -78.degree. C. for 15 min and then at
-20.degree. C. for 2 h, before being recooled at -78.degree. C. and
again being treated with boron tribromide (1 M in dichloromethane,
0.10 mL, 0.10 mmol). The mixture was then kept at 5.degree. C. for
18 h and at room temperature for an additional 5 h. This process
was repeated once more and then the resulting mixture was
partitioned with 1 N HCl and ethyl acetate. The organic phase was
separated, washed (1 N HCl, brine), dried (Na.sub.2SO.sub.4) and
evaporated, and the residue obtained was purified by prep tlc
(THF-hexane, 2:1) to give an inseparable 1:1 mixture of the title
compounds (0.0032 g, 36%) as a nearly colourless solid:
[0161] .sup.1H NMR (acetone-d.sub.6, 400 MHz) .delta. 9.34 (dd,
J=11.8 Hz, 9.0 Hz, 1H), 9.08 (dd, J=11.6 Hz, 8.6 Hz, 1H), 7.94 (dd,
J=10.5 Hz, 7.9 Hz, 1H), 7.81-7.72 (m, 1H), 7.69-7.59 (m, 1H), 6.47
(dd, J=5.4 Hz, 3.2 Hz, 1H), 5.48-5.31 (m, 2H), 5.03-4.90 (m, 2H),
4.50-4.45 (m, 1H), 4.20-4.12 (m, 1H), 4.07-4.02 (m, 1H), 3.61 (m,
1H), 2.72 (s, 3H).
[0162] MS (ESI.sup.-) m/e 526 (M-H).sup.-.
[0163] HPLC: 98.0% (320 nm).
EXAMPLE 24
2,10-Difluoro-12,13-[1,2-.beta.-D-glucopyranosyl-6,7,12,13-tetrahydro(5,H)-
-indolo-[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione
[0164] 33
[0165] A mixture of
2,10-difluoro-12-(3,4,6-tri-O-benzyl-.beta.-D-glucopyr-
anosyl]-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-d-
ione (0.100 g, 0.126 mmol) and flame-dried, powdered 4A molecular
sieves (0.10 g) in dry pyridine (3 mL) was cooled to 0.degree. C.
under nitrogen for 15 min before methanesulfonyl chloride (0.012
mL, 0.15 mmol) was added. The mixture was stirred for 8 h at room
temperature and then it was recooled to 0.degree. C. and treated
with additional methanesulfonyl chloride (0.015 mL). The mixture
was then stirred at room temperature for 1 h. This sequence was
repeated every hour until all of the starting material was
consumed. The mixture was then diluted with ethyl acetate and
washed with 0.1 N HCl, saturated NaHCO.sub.3 and brine, prior to
drying and solvent evaporation. Purification of the residue by
flash chromatography (ethyl acetate-hexane, 3:7) afforded the
mesylate (0.080 g, 73%) as a yellow solid which was used directly
in the next step. The mesylate was taken up in anhydrous
dimethylformamide (1 mL) and dry diisopropylethylamine (0.050 mL,
0.29 mmol) was added. The mixture was heated to 110.degree. C. for
1 h and then was stirred at ambient temperature overnight. The
resulting mixture was diluted with ethyl acetate and THF, washed
with 1 N HCl and brine, dried and concentrated. The residue was
taken up in EtOH-THF-MeOH (3:3:1), 10% palladium
hydroxide-on-carbon (0.30 g) was added and the mixture was
hydrogenated in a Parr shaker at room temperature and 60 psi for 24
h. The mixture was then filtered through Celite and the filtercake
was washed with THF and methanol. The filtrate was concentrated in
vacuo and the residue was purified by flash chromatography
(chloroform-methanol, 9:1) to yield the title compound (0.020 g,
32% overall) as a yellow solid, mp>300.degree. C.:
[0166] IR (KBr) 3307 (br), 2926, 2876, 1745, 1710, 1697, 1619,
1575, 1466, 1438, 1349, 1322, 1174, 1117, 1070, 1025, 760
cm.sup.-1.
[0167] .sup.1H NMR (DMSO-d.sub.6, 500 MHz) .delta. 11.09 (s, 1H),
8.81 (d, J=10.8 Hz, 1H), 8.73 (dd, J=8.3, 6.4 Hz, 1H), 8.61 (dd,
J=8.4, 5.6 Hz, 1H), 7.87 (d, J=8.0 Hz, 1H), 7.34-7.27 (m, 2H), 6.84
(d, J=4.6 Hz, 1H), 6.80 (s, 1H), 5.64 (d, J=5.1 Hz, 1H), 5.15 (s,
1H), 4.40-4.33 (m, 2H), 3.70-3.68 (m, 1H), 3.59-3.58 (m, 1H),
3.52-3.48 (m, 1H), 3.37-3.25 (m, 1H). MS (ESI.sup.-) m/e 504
(M-H).sup.-.
EXAMPLE 25
3,9-Difluoro-12,13-[1,2-.beta.-D-galactopyranosyl]-6,7,12,13-tetrahydro(5H-
)-indolo-[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione
[0168] 34
[0169] To a stirred solution of
3,9,-difluoro-6-(4-tert-butylbenzyl)-12-(4-
-deoxy-4-fluoro-.beta.-D-glucopyranosyl)]-6,7,12,13-tetrahydro(5H)indolo[2-
,3-a]pyrrolo[3,4-c]carbazole-5,7-dione (2.22 g, 3.31 mmol) in
absolute ethanol (750 mL) was added 4.45 M KOH (75 mL) and the
resulting blood red solution was allowed to stir overnight at room
temperature. The reaction was acidified with conc. HCl (80 mL),
solid ammonium acetate (750 g, ) and absolute ethanol (350 mL) were
added and the reaction mixture was refluxed for 5 days. The
reaction mixture was then concentrated by approximately 2/3, cooled
to room temperature and ethyl acetate (3500 mL) and water (1500 mL)
were added. The organic layer was extracted with water
(4.times.1500 mL), saturated sodium bicarbonate (1.times.1500 mL),
water (1.times.1500 mL) and brine (1.times.1500 mL), dried over
Na.sub.2SO.sub.4, filtered and the solvent removed in vacuo. The
resulting residue was pre-adsorbed onto silica gel and applied to a
flash column packed in methylene chloride and eluted with a
methylene chloride-acetone gradient. Further purification on
Sephadex LH-20 in methanol gave the 0.0237 g of the title compound
(1.4%): 500 MHz COSY .sup.1H NMR (d.sub.6-DMSO) .delta. 11.04 (brs,
1H), 8.32 (dd, 1H, J 9.1, 2.8 Hz), 8.22 (dd, 1H, 1J=9.1, 2.6 Hz),
8.06 (dd, 1H, J=9.0, 4.4 Hz), 7.93 (dd, 1H, J=9.3, 4.3 Hz),
7.45-7.35 (m, 2H), 6.72 (brs, 1H, 1'H), 6.13 (brs, 1H, 3'OH), 5.38
(brs, 1H, 3'H), 4.77 (brs, 1H, 2'H), 4.73 (d, 1H, 4'OH, J=5.5 Hz),
4.73 (t, 1H, 6'OH, J=5.4 Hz), 4.28 (dd, 1H, 5'H, J=6.2, 6.1 Hz),
3.71 (brs, 1H, 4'H), 3.38-3.18 (m, 2H, 6'H, 6"H); MS (ESI.sup.-)
m/e 504 (M-H).sup.-.
EXAMPLE 26
3,9-Difluoro-12,13-[1,6-(4-deoxy-4-fluoro-.beta.-D-glucopyranosyl)]-6,7,12-
,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione
[0170] 35
[0171] To a stirred solution of
3,9,-difluoro-12-(4-deoxy-4-fluoro-.beta.--
D-glucopyranosyl)]-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carb-
azole-5,7-dione (0.0100 g, 0.019 mmol) and triphenylphosphine
(0.010 g, 0.038 mmol) in anhydrous THF (200 .mu.L) was added
diisopropyl azodicarboxylate (7.5 .mu.L, 0.038 mmol). The resulting
blood red solution was stirred at room temperature for 3 h, after
which additional diisopropyl azodicarboxylate (3 .mu.L, 0.015 mmol)
was added. The reaction was stirred at room temperature for an
additional 30 minutes, then quenched with methanol (1 mL) and the
solvent removed in vacuo. Purification by flash column
chromatography on silica gel using an acetone-hexane gradient,
followed by purification on Sephadex LH-20 in methanol, gave 0.0041
g (42.5%) of the title compound: 500 MHz COSY .sup.1H NMR
(d.sub.6-DMSO) .delta. 8.97 (dd, 1H, J =9.7, 2.5 Hz), 8.88 dd, (1H,
J=9.7, 2.5 Hz), 8.34 (dd, 1H, J=9.2, 4.5 Hz), 8.08 (dd, 1H, J=9.0,
4.2 Hz), 7.54-7.45 (m, 2H), 6.20 (d, 1H, 1'H, J=7.80 Hz), 5.11
(brd, 1H, 6'H, J=12 Hz), 4.91 (dd, 1H, 4'H, J.sub.H-F=50.6 Hz,
J.sub.3',4'=5.4 Hz), 4.68 (brs, 1H, 5'H), 4.71-4.64 (m, 1H, 6"H),
4.60-4.45 (m, 1H, 2'H, J.sub.2,3=5.6 Hz), 3.99 (brddd, 1H, 3'H,
J.sub.H-F=28 Hz, J.sub.3,4 =5.5 Hz, J.sub.2,3=5.6 Hz); MS
(ESI.sup.-) m/e 506 (M-H).sup.-. Confirmed by X-ray
crystallography.
EXAMPLE 27 and 28
3,9-Difluoro-12,13-[1,6-.beta.-D-glucopyranosyl)]-6,7,12,13-tetrahydro(5H)-
indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione (1) and
3,9-Difluoro-12,13-[1,2-.beta.-D-glucopyranosyl]-6,7,12,13-tetrahydro(5H)-
-indolo-[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione (2)
[0172] 36
[0173] To a stirred solution of
3,9,-difluoro-12-.beta.-D-glucopyranosyl-6-
,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione
(0.240 g, 0.459 mmol) and triphenylphosphine (0.263 g, 1.00 mmol)
in anhydrous THF (10 mL) was added diisoproply azodicarboxylate
(200 .mu.L, 1.01 mmol) and the resulting blood red solution was
allowed to stir at room temperature for 2 h. The reaction was then
quenched with water (5 drops) and the solvent removed in vacuo.
Partial purification by flash column chromatography on silica gel
(acetone-hexane) afforded 2 components which were individually
repurified on Sephadex LH-20 (methanol) to give the 2 pure title
compounds. The first was identified as
3,9-difluoro-12,13-[1,6-.beta.-D-glucopyranosyl)]-6,7,12,13-tetrahydro-
(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione (0.019 g, 8.2%):
500 MHz COSY .sup.1H NMR (d.sub.6-DMSO) .delta. 8.87 (dd, 1H,
J=9.8, 2.6 Hz), 8.79 (dd, 1H, J=9.7, 2.6), 8.30 (dd, 1H, J=9.1, 4.3
Hz), 7.88 (dd, 1H, J=9.1, 4.3 Hz), 7.52-7.39 (m, 2H), 6.03 (d, 1H,
1'-H, J=7.8 Hz), 5.60 (br s, 1H, 4'-OH), 5.50 (br s, 1H, 3'-OH),
4.81 (br d, 1H, 6'-H, J=12.0 Hz), 4.60 (dd, 1H, 6'-H, J=12.2 Hz),
4.40-4.28 (m, 2H, 2'-H, 5'-H), 3.78 (br d, 1H, 4'-H), 3.70-3.62 (m,
1H, 3'-H);
[0174] MS (ESI.sup.-) m/e 504 (M-H).sup.-.
[0175] The second was identified as
3,9-difluoro-12,13-[1,2.beta.-D-glucop-
yranosyl]-6,7,12,13-tetrahydro(5H)-indolo-[2,3-a]pyrrolo[3,4-c]carbazole-5-
,7-dione (0.019 g, 8.2%):
[0176] 500 MHz COSY .sup.1H NMR (d.sub.6-DMSO) .delta. 11.01 (br s,
1H), 8.84 (dd, 1H, J=9.4, 4.6 Hz), 8.32 (dd, 1H, J=9.0, 2.8 Hz),
8.20 (dd, 1H, J=9.1, 2.7 Hz), 7.89 (dd, 1H, J=9.0, 4.5 Hz), 7.42
(m, 1H), 7.35 (m, 1H), 6.72 (s, 1H, 1'-H), 6.57 (d, 1H, 3'-OH,
J=4.80 Hz), 5.45 (d, 1H, 4'-OH, J=5.10 Hz), 5.06 (br s, 1H, 2'-H),
4.32-4.22 (m, 2H, 3'-H, 6'-H), 3.62-3.57 (m, 1H, 5'-H), 3.52-3.45
(m, 1H, 4'-H), 3.40 (d, 1H, 6'-H); MS (ESI.sup.-) m/e 504
(M-H).sup.-.
Topoisomerase I Activity (In Vitro)
[0177] Topoisomerase I activity was measured as described below:
The procedure for assaying compound-induced, topoisomerase
I-mediated single strand breaks in DNA was essentially that
described by Hsiang, et al., (J. Biol Chem. 1985, 260,
14873-14878). Samples dissolved in 100% DMSO as either 10 .mu.M or
10 mg/ml solutions, unless otherwise stated, were diluted in
Tris-EDTA buffer. Marine bacteriophage PM2 DNA (Boehringer
Mannheim) was also diluted in Tris-EDTA buffer to a concentration
of 0.02 .mu.g/.mu.l. Different dilutions of the compound being
evaluated were mixed with diluted DNA and this mixture was added to
1000 unit (one unit of enzyme activity is defined as the amount
capable of relaxing 100 ng of supercoiled DNA in approximately 30
minutes at 37.degree. C.) aliquots of purified human topoisomerase
I (Topogen) in 2.times. reaction buffer to start the reaction. The
compound-DNA-enzyme mixture was incubated for 30 minutes at
37.degree. C. before stopping the reaction with warn stop buffer
containing sodium dodecyl sulfate and proteinase K (Sigma). These
mixtures were allowed to incubate at 37.degree. C. for another 10
minutes, at which time the mixtures were removed from the waterbath
and extracted with a 24:1 mixture of chloroform/isoamyl alcohol.
Following centrifugation, aliquots of the aqueous phases were
placed in wells of a 0.9% agarose (SeaKem) gel in Tris-borate
buffer containing 0.5 .mu.g/ml of ethidium bromide and subjected to
electrophoresis for 15 hours to separate the different topological
isomers and nicked and broken DNAs. After destaining the gel in
water, the ethidium bromide stained DNA reaction products were
visualized by exposing the gel to UV irradiation. Negatives of the
photographs of the irradiated gels were scanned with a densitometer
and areas under the peaks were calculated in order to obtain
percent single strand DNA break formation for each sample. A median
effective concentration (EC.sub.50) was obtained for each compound
by interpolation between points of the resulting dose-effect curve
which defines the potency of the compound for its effect in
inducing topoisomerase I-mediated single strand breaks in DNA.
[0178] The topoisomerase I activity for certain compounds of the
present invention is shown below in Table I.
1 TABLE I Example No. EC.sub.50 (.mu.M) 13 8.2 16 2.9 21 0.29 22
0.32 23 2.1 3 0.35 4 5.6 8 0.62 9 >100 6 >100 5 0.68 7
>100 24 >100 10 0.45 11 3.3 25 0.110 26 0.028 27 0.036 28
0.120
In Vitro Cell-Based Cytotoxicity Activity
[0179] The proliferation inhibition activity against murine P388
cell line was measured as follows. Evaluation of a soluble
tetrazolium/formazan assay for cell growth and drug sensitivity in
culture, using human and other tumor cell lines, was done according
to the procedure described in Cancer Res. 1988, 48, 4827-4833.
Cells were plated at 4000 cells/well in 96 well microtiter plates
and 24 h later drugs were added and serially diluted. The cells
were incubated at 37.degree. C. for 72 h, at which time a
tetrazolium dye, XTT, containing phenazine methosulfate was added.
A dehydrogenase enzyme in live cells reduced the XTT to a form that
absorbs light at 450 ram, which could be quantitated
spectrophotometrically. The greater the absorbance the greater the
number of live cells. The results are expressed as an IC.sub.50,
which is the drug concentration required to inhibit cell
proliferation (i.e., absorbance at 450 nm) to 50% of that of
untreated control cells. The results for certain compounds of the
present invention are shown in Table II.
2 TABLE II Example No. EC.sub.50 (.mu.M) 13 2.8 16 3.3 21 0.14 22
0.086 23 0.24 3 0.05 4 0.07 8 0.026 9 0.021 6 7.7 5 0.023 7 0.46 24
1.15 10 0.13 11 0.021 25 0.0142 26 0.0030 27 0.0511 28 0.3468
* * * * *